T-cell death-inducing epitopes

ABSTRACT

Cell death-inducing epitopes and polypeptides containing same. Also disclosed are compounds for inducing death of activated T-cells, a method of producing antibodies to the epitopes, a method of identifying compounds that bind to the epitopes, a method of inducing death of activated T-cells, and pharmaceutical compositions containing the compounds.

RELATED APPLICATION

This application claims priority to U.S. Provisional Application Ser.No. 60/570,161, filed on May 11, 2004, the contents of which areincorporated by reference in its entirety.

BACKGROUND

Control of unwanted immune responses is critical in treating autoimmunediseases, transplant rejection, allergic diseases, and T-cell-derivedcancers. The activity of overly aggressive T-cells can be contained byimmunosuppression or by induction of immunological tolerance. Apoptosisis believed to be involved in maintaining proper functions of the immunesystem and removing unwanted cells, such as overly aggressive T-cells(Kabelitz et al. (1993) Immunol Today 14, 338-340; and Raff (1992)Nature 356, 397-399).

SUMMARY

This invention relates to T-cell death-inducing epitopes. The epitopescan be used for, among others, selecting compounds that bind to theepitopes. Such compounds are useful in treating diseases involvingoverly aggressive T-cells. Examples of such diseases include autoimmunediseases, transplant rejection, allergic diseases, and T-cell-derivedcancers.

In one aspect, the invention features a three-dimensional conformationof an isolated epitope. Binding of a ligand to the epitope on activatedT-cells induces death of the cells. Such an epitope is represented by:

-   -   (1) X₁-X₂-X₃-X₄-X₅ (SEQ ID NO:1), where        -   X₁ is Tyr, Trp, His, or Met;        -   X₂ is Asp;        -   X₃ is Ser, Phe, Pro, Glu, or His;        -   X₄ is any amino acid that naturally occurring in animals;            and        -   X₅ is Pro, Tyr, His, or Trp;    -   (2) X₆-X₇-X₈-X₉-X₁₀ (SEQ ID NO:2), where        -   X₆ is Asp;        -   X₇ is Tyr, Met, Asn, Trp, or Phe;        -   X₈ is Phe or Leu;        -   X₉ is Pro; and        -   X₁₀ is Glu; or    -   (3) X₁₁-X₁₂-X₁₃-X₁₄ (SEQ ID NO:3), where        -   X₁₁ is Pro;        -   X₁₂ is Met;        -   X₁₃ is Glu or Ser; and        -   X₁₄ is Ile.

Any of those epitopes described above can be, e.g., a polypeptide, aninteracting region of two polypeptides, a carbohydrate moiety, aglycoprotein, or any conformational, functional equivalent thereof.

In another aspect, the invention features an isolated polypeptidecontaining X₁-X₂-X₃-X₄-X₅, X₆-X₇-X₈-X₉-X₁₀, or X₁₁-X₁₂-X₁₃-X₁₄. Bindingof a ligand to the polypeptide on activated T-cells induces death of thecells. In one embodiment, the polypeptide contains 4 to 400 amino acids(e.g., any integer between 4 and 400, inclusive). For example, thepolypeptide can be X₁-X₂-X₃-X₄-X₅ (SEQ ID NO:1), X₆-X₇-X₈-X₉-X₁₀ (SEQ IDNO:2), X₁₁-X₁₂-X₁₃-X₁₄ (SEQ ID NO:3), or any of SEQ ID NOs:4, 6-18, and20-22.

An “isolated epitope” or “isolated polypeptide” refers to an epitope orpolypeptide substantially free from naturally associated molecules,i.e., it is at least 75% (e.g., any number between 75% and 100%,inclusive) pure by dry weight. Purity can be measured by any appropriatestandard method, for example, by column chromatography, polyacrylamidegel electrophoresis, or HPLC analysis. An isolated epitope orpolypeptide of the invention can be purified from a natural source,produced by recombinant DNA techniques, or by chemical methods.

In still another aspect, the invention features a novel compound thatbinds to one of the above-described epitopes. The compound can be anykind of molecule, including antibodies such as monoclonal antibodies. Acompound of the invention can be used for detecting an epitope of theinvention and for inducing death of activated T-cells. Also within thescope of the invention is a method of producing antibodies. The methodinvolves administering to a subject an effective amount of one of theabove-described epitopes (e.g., polypeptides). The antibodies can beused for detecting an epitope of the invention or for inducing death ofactivated T-cells.

The invention also features a method of identifying a candidate compound(e.g., a monoclonal antibody) for inducing death of activated T-cells.The method involves contacting a test compound with an epitope of theinvention and determining whether the test compound binds to theepitope. If the test compound binds to the epitope, it is a candidatefor inducing death of activated T-cells.

The invention further features a method of inducing death of activatedT-cells by contacting activated T-cells with a compound of theinvention.

In yet another aspect, the invention features a pharmaceuticalcomposition containing a pharmaceutically acceptable carrier and (1) anepitope of the invention such as a polypeptide, or (2) a compound thatbinds to the epitope.

The invention provides compositions and methods for treating diseasesinvolving overly aggressive T-cells such as autoimmune diseases,transplant rejection, allergic diseases, and T-cell-derived cancers. Thedetails of one or more embodiments of the invention are set forth in theaccompanying description below. Other features, objects, and advantagesof the invention will be apparent from the detailed description.

DETAILED DESCRIPTION

This invention is based on the unexpected discovery that activatedT-cells can be induced to undergo apoptosis and be depleted byengagement of new T-cell death-inducing epitopes. Depletion of activatedT-cells are particularly useful for treating conditions associated withan excessive or unwanted T-cell-mediated immune response or T-cellproliferation. For example, depletion of activated T-cells can result inreduction or elimination of undesirable T-cell activity or proliferationrelated to autoimmune diseases, transplant rejection, allergic diseases,or T-cell-derived cancers.

Accordingly, the invention features a three-dimensional conformation ofan isolated epitope. Binding of a ligand to the epitope on activatedT-cells induces death of the cells. The epitope is represented byX₁-X₂-X₃-X₄-X₅, X₆-X₇-X₈-X₉-X₁₀, or X₁₁-X₁₂-X₁₃-X₁₄. Thethree-dimensional conformation of X₁-X₂-X₃-X₄-X₅, X₆-X₇-X₈-X₉-X₁₀, orX₁₁-X₁₂-X₁₃-X₁₄ can be determined, e.g., using computer modelingprograms as described in Duggan et al., (1995) J Med Chem. 38:3332-41and Toogood (2002) J Med Chem. 45: 1543-57. Epitopes of conformational,functional equivalence can be designed in accordance with thethree-dimensional conformation of X₁-X₂-X₃-X₄-X₅, X₆-X₇-X₈-X₉-X₁₀, orX₁₁-X₁₂-X₁₃-X₁₄, prepared using methods known in the art, and tested fortheir abilities to be involved in induction of death of activatedT-cells by methods such as that described in the example below. See,e.g., Barbas et al. (2001) Phage display. A laboratory manual. CSHLPress; Parmley et al. (1998) Gene 73, 305-318; Scott et al. (1990)Science 249, 386-390; U.S. Patent Application No. 20030049252 A1; WO03/013603 A1; Osborne (1996) Curr Opin Immunol 8:245-248; Lin et al.(1997) J. Immunol. 158, 598-603; Zhang et al. (1995) Nature 377,348-350; Lai et al. (1995) Eur J Immunol 25, 3243-3248; Mollereau et al.(1996) J Immunol 156, 3184-3190; and Gribben et al. (1995) Proc NatlAcad Sci USA 92, 811-815.

As used herein, an “activated T-cell” is a T-cell having a higherfrequency, rate, or extent of proliferation than that of a non-activatedT-cell. “Death” of a cell includes programmed cell death, i.e.,apoptosis. “Induction of cell death” by an agent occurs when apopulation of cells treated with the agent exhibits a higher death ratecompared to an untreated cell population. For example, the percentage ofin vitro activated T-cells undergoing apoptosis is about doubled whentreated with monoclonal antibodies m128-9F9, m152-15A7, or m166-43B6compared to that of untreated cells, as determined by annexin V stainingand FACS analysis (see the example below).

The invention also features an isolated polypeptide containingX₁-X₂-X₃-X₄-X₅, X₆-X₇-X₈-X₉-X₁₀, or X₁₁-X₁₂-X₁₃-X₁₄. The polypeptide canbe used for identifying compounds that induce death of activatedT-cells. Binding of such a compound to the polypeptide expressed on thesurface of activated T-cells induces cell death. Further, freepolypeptides (i.e., those not expressed on the cell surface) can inhibitunwanted cell death by competing for endogenous death-inducing ligandswith the cell-surface polypeptides. The length or sequence of thepolypeptide may vary for these uses. A polypeptide of the invention canbe obtained, e.g., as an isolated T-cell surface protein, a syntheticpolypeptide, or a recombinant polypeptide. To prepare a recombinantpolypeptide, a nucleic acid encoding it can be linked to another nucleicacid encoding a fusion partner, e.g., Glutathione-S-Transferase (GST),6×-His epitope tag, or M13 Gene 3 protein. The resultant fusion nucleicacid expresses in suitable host cells a fusion protein that can beisolated by standard methods. The isolated fusion protein can be furthertreated, e.g., by enzymatic digestion, to remove the fusion partner andobtain the recombinant polypeptide of this invention.

An epitope of the invention or a polypeptide of the invention can beused to generate antibodies in animals (for production of antibodies) orhumans (for treatment of diseases). Methods of making monoclonal andpolyclonal antibodies and fragments thereof in animals are known in theart. See, for example, Harlow and Lane, (1988)

Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y. Theterm “antibody” includes intact molecules as well as fragments thereof,such as Fab, F(ab′)₂, Fv, scFv (single chain antibody), and dAb (domainantibody; Ward, et. al. (1989) Nature, 341, 544). These antibodies canbe used for detecting the epitope, e.g., in identifying a compound thatbinds to the epitope (see below). The antibodies that are capable ofinducing death of activated T-cells are also useful for treatingdiseases such as autoimmune diseases, transplant rejection, allergicdiseases, or T-cell-derived cancers. In general, an epitope of theinvention, e.g., a polypeptide, can be coupled to a carrier protein,such as KLH, mixed with an adjuvant, and injected into a host animal.Antibodies produced in that animal can then be purified by peptideaffinity chromatography. Commonly employed host animals include rabbits,mice, guinea pigs, and rats. Various adjuvants that can be used toincrease the immunological response depend on the host species andinclude Freund's adjuvant (complete and incomplete), mineral gels suchas aluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanin, and dinitrophenol. Useful human adjuvants include BCG(bacille Calmette-Guerin) and Corynebacterium parvum.

Polyclonal antibodies, heterogeneous populations of antibody molecules,are present in the sera of the immunized subjects. Monoclonalantibodies, homogeneous populations of antibodies to a particularantigen, can be prepared using standard hybridoma technology (see, forexample, Kohler et al. (1975) Nature 256, 495; Kohler et al. (1976) EurJ Immunol 6, 511; Kohler et al. (1976) Eur J Immunol 6, 292; andHammerling et al. (1981) Monoclonal Antibodies and T Cell Hybridomas,Elsevier, N.Y.). In particular, monoclonal antibodies can be obtained byany technique that provides for the production of antibody molecules bycontinuous cell lines in culture such as described in Kohler et al.(1975) Nature 256, 495 and U.S. Pat. No. 4,376,110; the human B-cellhybridoma technique (Kosbor et al. (1983) Immunol Today 4, 72; Cole etal. (1983) Proc Natl Acad Sci USA 80, 2026, and the EBV-hybridomatechnique (Cole et al. (1983) Monoclonal Antibodies and Cancer Therapy,Alan R. Liss, Inc., pp. 77-96). Such antibodies can be of anyimmunoglobulin class including IgG, IgM, IgE, IgA, IgD, and any subclassthereof. The hybridoma producing the monoclonal antibodies of theinvention may be cultivated in vitro or in vivo. The ability to producehigh titers of monoclonal antibodies in vivo makes it a particularlyuseful method of production.

In addition, techniques developed for the production of “chimericantibodies” can be used. See, e.g., Morrison et al. (1984) Proc NatlAcad Sci USA 81, 6851; Neuberger et al. (1984) Nature 312, 604; andTakeda et al. (1984) Nature 314:452. A chimeric antibody is a moleculein which different portions are derived from different animal species,such as those having a variable region derived from a murine monoclonalantibody and a human immunoglobulin constant region. Alternatively,techniques described for the production of single chain antibodies (U.S.Pat. Nos. 4,946,778 and 4,704,692) can be adapted to produce a phagelibrary of single chain Fv antibodies. Single chain antibodies areformed by linking the heavy and light chain fragments of the Fv regionvia an amino acid bridge. Moreover, antibody fragments can be generatedby known techniques. For example, such fragments include, but are notlimited to, F(ab′)₂ fragments that can be produced by pepsin digestionof an antibody molecule, and Fab fragments that can be generated byreducing the disulfide bridges of F(ab′)₂ fragments. Antibodies can alsobe humanized by methods known in the art. For example, monoclonalantibodies with a desired binding specificity can be commerciallyhumanized (Scotgene, Scotland; and Oxford Molecular, Palo Alto, Calif.).Fully human antibodies, such as those expressed in transgenic animalsare also features of the invention (see, e.g., Green et al. (1994)Nature Genetics 7, 13; and U.S. Pat. Nos. 5,545,806 and 5,569,825).

The invention further features a novel compound that binds to an epitopeof the invention and induces death of activated T-cells. Such a compoundcan be designed, e.g., using computer modeling programs, according tothe three-dimensional conformation of the epitope, and synthesized usingmethods known in the art. It can also be identified by library screeningas described below.

The test compounds can be obtained using any of the numerous approachesin combinatorial library methods known in the art. Such librariesinclude: peptide libraries, peptoid libraries (libraries of moleculeshaving the functionalities of peptides, but with a novel, non-peptidebackbone that is resistant to enzymatic degradation), spatiallyaddressable parallel solid phase or solution phase libraries, syntheticlibraries obtained by deconvolution or affinity chromatographyselection, the “one-bead one-compound” libraries, and antibodylibraries. See, e.g., Zuckermann et al. (1994) J Med Chem 37, 2678-85;Lam (1997) Anticancer Drug Des 12, 145; Lam et al. (1991) Nature 354,82; Houghten et al. (1991) Nature 354, 84; and Songyang et al. (1993)Cell 72, 767.

Examples of methods for the synthesis of molecular libraries can befound in the art, for example, in: DeWitt et al. (1993) PNAS USA 90,6909; Erb et al. (1994) PNAS USA 91, 11422; Zuckermann et al. (1994) JMed Chem 37, 2678; Cho et al. (1993) Science 261, 1303; Carrell et al.(1994) Angew Chem Int Ed Engl 33, 2059; Carell et al. (1994) Angew Chemhit Ed Engl 33, 2061; and Gallop et al. (1994) J Med Chem 37,1233.

Libraries of compounds may be presented in solution (e.g., Houghten(1992) Biotechniques 13, 412-421), or on beads (Lam (1991) Nature 354,82-84), chips (Fodor (1993) Nature 364, 555-556), bacteria (U.S. Pat.No. 5,223,409), spores (U.S. Pat. No. 5,223,409), plasmids (Cull et al.(1992) PNAS USA 89, 1865-1869), or phages (Scott and Smith (1990)Science 249, 386-390; Devlin (1990) Science 249, 404-406; Cwirla et al.(1990) PNAS USA 87, 6378-6382; Felici (1991) J Mol Biol 222, 301-310;and U.S. Pat. No. 5,223,409).

To identify a candidate compound for inducing death of activatedT-cells, an epitope of the invention is contacted with a test compound,and the binding of the compound to the epitope is evaluated. If thecompound binds to the epitope, it is a candidate for inducing death ofactivated T-cells.

The screening assay can be conducted in a variety of ways. For example,one method involves anchoring the epitope (or an epitope-containingmolecule, e.g., a polypeptide or a fusion protein) or the test compoundonto a solid phase and detecting an epitope-test compound complex formedon the solid phase at the end of the reaction. In practice, microtiterplates may conveniently be utilized as the solid phase. The anchorcomponent may be immobilized by non-covalent or covalent attachments.Non-covalent attachment may be accomplished by simply coating the solidsurface with a solution of the anchor component and drying the plates.Alternatively, an immobilized antibody (e.g., a monoclonal antibody)specific for the anchor component may be used to immobilize the anchorcomponent to the solid surface. The non-anchor component is added to thesolid surface coated with the anchor component. After the reaction iscomplete, unbound fraction of the non-anchor components is removed(e.g., by washing) under conditions such that any complexes formedremain immobilized on the solid surface. Detection of these complexescan be accomplished in a number of ways. Where the non-anchor componentis pre-labeled, detection of the label immobilized on the solid surfaceindicates that complexes were formed. Where the non-anchor component isnot pre-labeled, an indirect label can be used to detect complexesformed on the surface, e.g., using an antibody specific for thenon-anchor component (the antibody, in turn, may be directly labeled orindirectly labeled with a labeled anti-Ig antibody).

Alternatively, the reaction can be conducted in a liquid phase. Thecomplexes are separated from unbound components, e.g., using animmobilized antibody specific for the epitope (or the epitope-containingmolecule) or the test compound. The complexes are then detected, e.g.,using a labeled antibody specific for the other component.

The candidate compound can be validated by ascertaining its ability toinduce death of activated T-cells, using the method described in theexample below, or any other method know in the art. The validatedcompound can be used for inducing death of activated T-cells and fortreating diseases such as autoimmune diseases, transplant rejection,allergic diseases, or T-cell-derived cancers.

The invention provides a method of inducing death of activated T-cells,e.g., by contacting activated T-cells with a compound of the inventionin vitro, or by administering to a subject in need thereof an effectiveamount of a compound of the invention. Subjects to be treated can beidentified as having or being at risk for acquiring a conditioncharacterized by an excessive or unwanted T-cell-mediated immuneresponse, e.g., patients suffering from autoimmune diseases, transplantrejection, allergic diseases, or T-cell-derived cancers. This method canbe performed alone or in conjunction with other drugs or therapy.

The term “treating” is defined as administration of a composition to asubject with the purpose to cure, alleviate, relieve, remedy, prevent,or ameliorate a disorder, the symptom of the disorder, the disease statesecondary to the disorder, or the predisposition toward the disorder. An“effective amount” is an amount of the composition that is capable ofproducing a medically desirable result, e.g., as described above, in atreated subject.

Exemplary diseases to be treated include, but are not limited to,diabetes mellitus, arthritis (including rheumatoid arthritis, juvenilerheumatoid arthritis, osteoarthritis, and psoriatic arthritis), multiplesclerosis, encephalomyelitis, myasthenia gravis, systemic lupuserythematosis, autoimmune thyroiditis, dermatitis (including atopicdermatitis and eczematous dermatitis), psoriasis, Sjogren's Syndrome,Crohn's disease, aphthous ulcer, iritis, conjunctivitis,keratoconjunctivitis, type I diabetes, inflammatory bowel diseases,ulcerative colitis, asthma, allergic asthma, cutaneous lupuserythematosus, scleroderma, vaginitis, proctitis, drug eruptions,leprosy reversal reactions, erythema nodosum leprosum, autoimmuneuveitis, allergic encephalomyelitis, acute necrotizing hemorrhagicencephalopathy, idiopathic bilateral progressive sensorineural hearingloss, aplastic anemia, pure red cell anemia, idiopathicthrombocytopenia, polychondritis, Wegener's granulomatosis, chronicactive hepatitis, Stevens-Johnson syndrome, idiopathic sprue, lichenplanus, Graves' disease, sarcoidosis, primary biliary cirrhosis, uveitisposterior, interstitial lung fibrosis, graft-versus-host disease, casesof transplantation (including transplantation using allogeneic orxenogeneic tissues) such as bone marrow transplantation, livertransplantation, or the transplantation of any organ or tissue,allergies such as atopic allergy, AIDS, and T-cell neoplasms such asleukemias or lymphomas.

In one in vivo approach, a therapeutic composition (e.g., a compositioncontaining an epitope of the invention, a polypeptide of the invention,or a compound of the invention) is administered to the subject.Generally, the epitope, the polypeptide, or the compound is suspended ina pharmaceutically-acceptable carrier (e.g., physiological saline) andadministered orally or by intravenous infusion, or injected or implantedsubcutaneously, intramuscularly, intrathecally, intraperitoneally,intrarectally, intravaginally, intranasally, intragastrically,intratracheally, or intrapulmonarily.

The dosage required depends on the choice of the route ofadministration; the nature of the formulation; the nature of thesubject's illness; the subject's size, weight, surface area, age, andsex; other drugs being administered; and the judgment of the attendingphysician. Suitable dosages are in the range of 0.01-100.0 mg/kg. Widevariations in the needed dosage are to be expected in view of thevariety of compositions available and the different efficiencies ofvarious routes of administration. For example, oral administration wouldbe expected to require higher dosages than administration by intravenousinjection. Variations in these dosage levels can be adjusted usingstandard empirical routines for optimization as is well understood inthe art. Encapsulation of the composition in a suitable delivery vehicle(e.g., polymeric microparticles or implantable devices) may increase theefficiency of delivery, particularly for oral delivery.

Also within the scope of this invention is a pharmaceutical compositionthat contains a pharmaceutically acceptable carrier and an effectiveamount of a compound of the invention. The pharmaceutical compositioncan be used to treat diseases described above. The pharmaceuticallyacceptable carrier includes a solvent, a dispersion medium, a coating,an antibacterial and antifungal agent, and an isotonic and absorptiondelaying agent.

The pharmaceutical composition of the invention can be formulated intodosage forms for different administration routes utilizing conventionalmethods. For example, it can be formulated in a capsule, a gel seal, ora tablet for oral administration. Capsules can contain any standardpharmaceutically acceptable materials such as gelatin or cellulose.Tablets can be formulated in accordance with conventional procedures bycompressing mixtures of the composition with a solid carrier and alubricant. Examples of solid carriers include starch and sugarbentonite. The composition can also be administered in a form of a hardshell tablet or a capsule containing a binder, e.g., lactose ormannitol, a conventional filler, and a tableting agent. Thepharmaceutical composition can be administered via the parenteral route.Examples of parenteral dosage forms include aqueous solutions, isotonicsaline or 5% glucose of the active agent, or other well-knownpharmaceutically acceptable excipient. Cyclodextrins, or othersolubilizing agents well known to those familiar with the art, can beutilized as pharmaceutical excipients for delivery of the therapeuticagent.

The efficacy of a composition of this invention can be evaluated both invitro and in vivo. See, e.g., the examples below. Briefly, thecomposition can be tested for its ability to induce death of activatedT-cells in vitro. For in vivo studies, the composition can be injectedinto an animal (e.g., a mouse model) and its therapeutic effects arethen accessed. Based on the results, an appropriate dosage range andadministration route can be determined.

The specific example below is to be construed as merely illustrative,and not limitative of the remainder of the disclosure in any waywhatsoever. Without further elaboration, it is believed that one skilledin the art can, based on the description herein, utilize the presentinvention to its fullest extent. All publications recited herein arehereby incorporated by reference in their entirety.

Preparation of a Mouse Spleen Cell Suspension

Mouse spleen was immersed in 8 ml of Hank's balanced salt solution(HBSS), gently minced with a sterile cover slip, transferred to a 15 mlcentrifuge tube (Costar), and spun at 200× g for 5 minutes. Thesupernatant was discarded, and the cell pellet was resuspended in theresidual buffer by gently tapping the wall. The contaminating red bloodcells (RBC) were lysed by addition of 1 ml of RBC lysis buffer (0.6 MNH₄Cl, 0.17 M Tris-base, pH 7.65), followed by a 2 min incubation atroom temperature and rapid quenching with 9 ml of HBSS. The cells werepelleted at 200×g for 5 minutes, washed twice, and resuspended in RPMImedium. The concentration and viability of the cells in the mixture weredetermined with a hemocytometer (Cambridge Scientific Inc.) and Trypanblue exclusion.

Preparation of Anti-T-Cell, Apoptosis-Inducing Monoclonal Antibodies

T-cell apoptosis-inducing monoclonal antibodies were generated byimmunizing a mouse with Concanovalin A-activated human T-cells andscreened for their abilities to bind to activated human T-cells andsubsequently to induce T-cell apoptosis. The monoclonal antibodies wereprepared according to the well-known cell fusion methods of Kohler andMilstein ((1976) Euro J Immunol 6, 511-519) to produce a hybridomasecreting desired antibodies. Three hybridomas generated according tothese methods secreted monoclonal antibodies, designated m128-9F9,m152-15A7, and m166-43B6, respectively, that were able to induce T-cellapoptosis in vitro.

Concentrated culture supernatant of each hybridoma was spun at 20000×gfor 10 minutes, and the supernatant was diluted at a 1:1 ratio with thebinding buffer (0.1 M sodium acetate, pH 5.0). A protein G column(approximately 1 ml bed volume) was washed three times with 3-5 ml ofthe binding buffer. The cleared culture supernatant was loaded onto theprotein G column, and the flow-through was collected and reloaded to thecolumn. The column was then washed with 6-10 ml of the binding buffer,and the bound antibody was eluted from the column with 5 ml of theelution buffer (0.1 M glycine-HCl, pH 2.8). Each fraction contained 1 mlof the eluted antibody, and the eluted fraction was adjusted to theneutral pH by mixing each 1 ml fraction with 50 microliters of 1 MTris-HCl, pH 7.5. Fractions containing the antibody were pooled anddialyzed against 2 liters of PBS, pH 7.4 three times for three hours perdialysis. Protein concentrations in the antibody samples were determinedfollowing the procedure described by Bradford using the Bio-Rad ProteinAssay (BIO-RAD, Hercules, Calif.).

Induction of Death of Activated Human T-Cells by Monoclonal Antibodies

Activated T cells (see above) were resuspended to a final concentrationof 5×10⁵ cells/ml in RPMI medium containing 5 ng/ml of IL-2, and treatedwith control Ig, m128-9F9, m152-15A7, or m166-43B6.

It is well known that T-cell death-inducing antibodies can be used astherapeutic agents to treat T-cell-related diseases such astransplantation rejections, autoimmune diseases, and allergy. Threemonoclonal antibodies against human T-cells were generated, and thecapabilities of these monoclonal antibodies to induce apoptosis ofactivated human T-cells were examined. Culture supernatants containingmonoclonal antibodies secreted by hybridoma cell line m128-9F9,m152-15A7, or m166-43B6 were incubated with either non-activated humanT-cells (Day 0) or in vitro activated human T-cells (Day7) for 6 hours.Cells were stained with annexin V after incubation, and subjected toFACS analysis. CD3-positive cells were gated to ensure counting ofeither in vitro activated human T-cells or resting human T-cells. Theapoptotic cells were annexin V staining-positive. Table 1 summarizes thepercentage of apoptotic T-cells among all of the T-cells scanned.Unexpectedly, monoclonal antibodies secreted by hybridoma cell linesm128-9F9, m152-15A7, and m166-43B6 induced death of in vitro activatedhuman T-cells but did not affect non-activated human T-cells. Thiscapability of inducing apoptosis of activated T-cells yet sparing theresting T-cells is a unique feature of the apoptotic pathway and is adominating feature of therapeutic reagents targeting T-cell-mediateddiseases.

TABLE 1 Percentage of apoptotic T-cells Untreated Anti-myc m128-9F9Untreated Anti-myc m152-15A7 m166-43B6 Day 0 4.17 6.67 5.82 18.18 15.525.23 6.57 Day 7 12.63 13.36 28.71 24.18 23.08 51.66 49.44

Identification of T-Cell Death-Inducing Epitopes

In order to identify death-inducing epitopes recognized by monoclonalantibodies m128-9F9, m152-15A7, and m166-43B6, these monoclonalantibodies were used to screen for consensus binding sequences in apolypeptide library (Ph. D.-12™ Phage Display Peptide Library Kit, NewEngland Biolabs, Inc.). The library contained various 12-mer peptideslinked to the 406-aa M13 Gene 3 protein. 96-well microtiter plates werecoated with 50 μl/well antibodies at the concentration of 10 μg/ml in0.1 M NaHCO₃ (pH 8.6) coating buffer overnight at 4° C. After the wash,the plates were blocked by incubation with the blocking buffercontaining 0.1 M NaHCO₃ (pH 8.6), 5 mg/ml BSA, 0.02% NaN₃ (150 μl/well)for at least one hour at 4° C. Plates were then incubated with fusionproteins from the polypeptide library described above at variousconcentrations for one hour at room temperature. After the wash with0.5% Tween containing TBS, the bound fusion proteins were eluted with 1mg/ml BSA containing 0.2 M Glycine-HCl (pH 2.2) buffer and neutralizedwith 1 M Tris-HCl (pH 9.1). The amino acid sequences of eluted fusionproteins were then determined.

The polypeptide sequences bound by monoclonal antibody m128-9F9 areshown below:

SEQ ID NO: 4 WPEDSS YDS W P RG SEQ ID NO: 5     LD YDF L P ETEPSEQ ID NO: 6    TAT WDP D Y FSDS SEQ ID NO: 7   AETD YDP D H FTPGSEQ ID NO: 8  DARYS HDP A W PYG SEQ ID NO: 9   AGQK WDP E W PHSGSEQ ID NO: 10    EPN MDP N W ASPSG SEQ ID NO: 11    KSH YDE S W WYNGGSEQ ID NO: 12        YDH H W TNPPTQK SEQ ID NO: 13        YDH H WPRDDIAP

A consensus polypeptide sequence of X₁-X₂-X₃-X₄-X₅ was obtained, whereX₁=Y/W/H/M, X₂=D, X₃=S/F/P/E/H, X₄=any amino acid, and X₅=P/Y/H/W.

The polypeptide sequences bound by monoclonal antibody m166-43B6 areshown below:

SEQ ID NO: 14 QDTWYP DYFPE S SEQ ID NO: 15 SHTLLN DMFPE S SEQ ID NO: 16  SPLR DNFPE TLW SEQ ID NO: 17  ASPYM DNFPE EN SEQ ID NO: 18   QLVQDWLPE ESH SEQ ID NO: 19   YLDY DFLPE TEPP

A consensus polypeptide sequence of X₆-X₇-X₈-X₉-X₁₀ was obtained, whereX₆=D, X₇=Y/M/N/W/F, X₈=F or L, X₉=P, and X₁₀=E.

The polypeptide sequences bound by monoclonal antibody m152-15A7 areshown below:

SEQ ID NO: 20     YTPM PMEI SHSA SEQ ID NO: 21   MNDKYI PMSI SASEQ ID NO: 22 KIPHKTLV PMEI SEQ ID NO: 23     TDSA AMEI QTTQ

A consensus polypeptide sequence of X₁₁-X₁₂-X₁₃-X₁₄ was obtained, whereX₁₁=P/A, X₁₂=M, X₁₃=E/S, and X₁₄=I.

ELISA Assay of T-Cell Death-Inducing Epitopes Recognized by MonoclonalAntibodies

In order to identify the specificities of the death-inducing epitopesrecognized by the monoclonal antibodies described above, the sandwichELISA was conducted. Serial dilutions (from 0.0017 fmol to 17 fmol) ofthe epitope-containing polypeptides were incubated with monoclonalantibodies m128-9F9, m152-15A7, or m166-43B6 pre-coated on the ELISAplates to determine their binding affinities.

96-well microtiter plates were coated with 50 μl/well antibodies at theconcentration of 1 μg/ml overnight at 4° C. Plates were blocked byincubation with 0.25% of BSA in PBS (150 μd/well) for 1 hour at 37° C.Plates were then incubated with fusion proteins containing variouspolypeptides for 2 hours at room temperature. After being washed 4 timeswith PBS containing 0.05% of Tween 20 (PBST), plates were then incubatedwith antibodies specific for the fusion partner at 2 μg/ml for 1.5 hoursat room temperature. After incubation, plates were washed 4 times withPBST. 50 μl of 1 to 3000 times diluted specific goat anti-fusion partnerantibodies conjugated with alkaline phosphotase (AP) was then added toeach well, and the plates were incubated for 1 hour at 37° C. Enzymereaction was carried out by adding 50 ul of AP substrate solution (1 APsubstrate tablet dissolved in 5 ml of substrate buffer). The resultsconfirmed that all of the selected polypeptides bind specifically totheir corresponding antibodies used for selection.

Other Embodiments

All of the features disclosed in this specification may be combined inany combination. Each feature disclosed in this specification may bereplaced by an alternative feature serving the same, equivalent, orsimilar purpose. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features.

From the above description, one skilled in the art can easily ascertainthe essential characteristics of the present invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions. Thus, other embodiments are also within the scope of theinvention.

1-27. (canceled)
 28. A method of identifying a candidate compound forinducing death of activated T-cells, the method comprising contacting acompound with a polypeptide comprising X₁-X₂-X₃-X₄-X₅, wherein X₁ isTyr, Trp, His, or Met; X₂ is Asp; X₃ is Ser, Phe, Pro, Glu, or His; X₄is any amino acid; and X₅ is Pro, Tyr, His, or Trp, wherein binding ofthe compound to the polypeptide indicates that the compound is acandidate for inducing death of activated T-cells. 29-31. (canceled) 32.The method of claim 28, wherein the polypeptide is selected from thegroup consisting of SEQ ID NOs:4 and 6-13.
 33. (canceled)
 34. The methodof claim 28, wherein the compound is an antibody.
 35. The method ofclaim 34, wherein the antibody is a monoclonal antibody.
 36. A method ofproducing an antibody for inducing death of activated T-cells, themethod comprising administering to a subject an effective amount of apolypeptide comprising X₁-X₂-X₃-X₄-X₅, wherein X₁ is Tyr, Trp, His, orMet; X₂ is Asp; X₃ is Ser, Phe, Pro, Glu, or His; X₄ is any amino acid;and X₅ is Pro, Tyr, His, or Trp. 37-39. (canceled)
 40. The method ofclaim 36, wherein the polypeptide is selected from the group consistingof SEQ ID NOs:4 and 6-13.
 41. (canceled)
 42. The method of claim 36,wherein the antibody is a monoclonal antibody.
 43. A method ofidentifying a candidate compound for inducing death of activatedT-cells, the method comprising contacting a compound with a polypeptidecomprising X₆-X₇-X₈-X₉-X₁₀, wherein X₆ is Asp; X₇ is Tyr, Met, Asn, Trp,or Phe; X₈ is Phe or Leu; X₉ is Pro; and X₁₀ is Glu, wherein binding ofthe compound to the polypeptide indicates that the compound is acandidate for inducing death of activated T-cells. 44-46. (canceled) 47.The method of claim 43, wherein the polypeptide is selected from thegroup consisting of SEQ ID NOs:14-18.
 48. (canceled)
 49. The method ofclaim 43, wherein the compound is an antibody.
 50. (canceled)
 51. Amethod of producing an antibody for inducing death of activated T-cells,the method comprising administering to a subject an effective amount ofa polypeptide comprising X₆-X₇-X₈-X₉-X₁₀, wherein X₆ is Asp; X₇ is Tyr,Met, Asn, Trp, or Phe; X₈ is Phe or Leu; X₉ is Pro; and X₁₀ is Glu.52-54. (canceled)
 55. The method of claim 51, wherein the polypeptide isselected from the group consisting of SEQ ID NOs:14-18.
 56. (canceled)57. The method of claim 51, wherein the antibody is a monoclonalantibody.
 58. A method of identifying a candidate compound for inducingdeath of activated T-cells, the method comprising contacting a compoundwith a polypeptide comprising X₁₁-X₁₂-X₁₃-X₁₄, wherein X₁₁ is Pro; X₁₂is Met; X₁₃ is Glu or Ser; and X₁₄ is Ile, wherein binding of thecompound to the polypeptide indicates that the compound is a candidatefor inducing death of activated T-cells. 59-61. (canceled)
 62. Themethod of claim 58, wherein the polypeptide is selected from the groupconsisting of SEQ ID NOs:20-22.
 63. (canceled)
 64. The method of claim58, wherein the compound is an antibody.
 65. The method of claim 64,wherein the antibody is a monoclonal antibody.
 66. A method of producingan antibody for inducing death of activated T-cells, the methodcomprising administering to a subject an effective amount of apolypeptide comprising X₁₁-X₁₂-X₁₃X₁₄, wherein X₁₁ is Pro; X₁₂ is Met;X₁₃ is Glu or Ser; and X₁₄ is Ile. 67-69. (canceled)
 70. The method ofclaim 66, wherein the polypeptide is selected from the group consistingof SEQ ID NOs:20-22.
 71. (canceled)
 72. The method of claim 66, whereinthe antibody is a monoclonal antibody.